Activation of a caspase-3-independent mode of cell death associated with lysosomal destabilization in cultured human retinal pigment epithelial cells (ARPE-19) exposed to 7beta-hydroxycholesterol.

PURPOSE: To characterize the possible cytotoxic effects of oxysterols (7beta-hydroxycholesterol (7beta-OH), 25-hydroxycholesterol (25-OH)) in human retinal pigment epithelial cells (ARPE-19) and to detail the relationships between some of these effects. METHODS: ARPE-19 cells were treated with 7beta-OH and 25-OH. Cell viability was measured with the MTT assay. Membrane permeability, mitochondrial potential, and lysosomal integrity were measured by flow cytometry with propidium iodide, DiOC6(3), and acridine orange, respectively. Cell death was characterized by staining with Hoechst 33342, transmission electron microscopy, and analysis of the DNA fragmentation pattern. Caspase activity was examined with fluorochrome-labeled inhibitors of caspases (FLICA) and Western blotting. Immunofluorescence staining was used to visualize the cellular distribution of cytochrome c (Cyt-c) and apoptosis-inducing factor (AIF). The effect of the cathepsin inhibitor (z-FA-fmk) on oxysterol-induced cell death was evaluated. RESULTS: Cell viability of ARPE-19 cells was decreased with 7beta-OH, whereas 25-OH had no cytotoxic effects. Loss of mitochondrial potential and lysosomal destabilization was associated with 7beta-OH-induced cell death, few morphologically apoptotic cells were identified, and no internucleosomal DNA fragmentation was found. Slight caspase activation was detected with FLICA, and no caspase-3 activation was revealed. A pronounced relocalization of Cyt-c and AIF was observed. Noteworthy, z-FA-fmk was able to prevent cell death. CONCLUSION: 7beta-OH induced a caspase-3-independent mode of cell death associated with lysosomal destabilization, which could play a key role in the signaling pathways leading to cell death, as shown by the ability of z-FA-fmk to counteract the cytotoxic effects of 7beta-OH.

Comparison of face-down and seated position after idiopathic macular hole surgery: a randomized clinical trial.

PURPOSE: To compare two therapeutic modalities on anatomic and functional results after idiopathic macular hole (MH) surgery: seated vs face-down position. DESIGN: Multicenter, prospective, randomized trial. SETTING: University Hospital Dijon and University Hospital Nancy. PATIENTS: One hundred and forty-four patients (150 eyes) were enrolled and randomly separated into two groups for postoperative position: for the 72 eyes in the P0 group and the 78 eyes in the P1 group, the patients were asked to keep the seated (P0 group) and the face-down position (P1 group) after the idiopathic MH surgery. INTERVENTION: All patients underwent a complete vitrectomy with a fluid-air exchange and an intraocular gas tamponade. After the surgery, patients were asked to keep one of the two randomly chosen positions for five days. MAIN OUTCOME MEASURES: Best-corrected visual acuity (VA), fundus examination, and macular optical coherence tomography were performed before and six months after surgery. RESULTS: The overall anatomic success rate was 92.7%. The idiopathic MH sealed in 63 of 72 P0 eyes (87.5%) and 76 of 78 P1 eyes (97.4%) (P = .027). The mean VA increased from 0.86 to 0.61 logMAR (0.88 to 0.61 in P0 and 0.84 to 0.60 in P1). However, in a post hoc analysis based on the size of the idiopathic MH, the success rate in idiopathic MHs smaller than 400 microm was not influenced by the postoperative position (P = .47). CONCLUSIONS: A face-down postoperative position is highly recommended in holes larger than 400 microm. The size of the idiopathic MH seems to be an important factor affecting outcome.

Incidence of retinal detachment after macular surgery: a retrospective study of 634 cases.

AIMS: To evaluate the rate of retinal detachment after macular surgery. METHODS: A retrospective non-randomised study of 634 macular surgery procedures was undertaken in two academic centres. Idiopathic macular hole (IMH) surgery (n = 272) and epiretinal membrane (ERM) surgery (n = 362) were performed between 2000 and 2003. We noted the anatomical and functional results of these procedures and we studied serious complications excluding cataract. RESULTS: Minimum follow up was 1 year. No retinal detachment (RD) occurred in patients presenting with an intraoperative or preoperative successfully treated retinal break (RB). The rate of RD occurring after IMH surgery was higher than after the ERM surgical procedure (6.6% vs 2.5%, p = 0.02). The rate of RD was higher in patients presenting with stage 2 and 3 IMH than with stage 4 IMH. However, lens status as well as preoperatively treated RD did not influence the rate of RD after macular surgery. CONCLUSION: Retinal detachment remains the most common serious complication of macular surgery. Surgical detachment of the posterior vitreous face and associated peripheral retina anomaly seem to increase the rate of this complication. Careful examination of the peripheral retina is a key issue in preventing retinal detachment occurring after macular surgery.

Oxysterols induced inflammation and oxidation in primary porcine retinal pigment epithelial cells.

PURPOSE: Aging is associated with an accumulation of cholesterol esters in the Bruch membrane. Cholesterol esters are prone to undergo oxidation and generate oxysterols that have cytotoxic and proinflammatory properties. We investigated the effects of three oxysterols on mitochondrial dysfunctions, inflammation, and oxidative stress in primary cultures of porcine retinal pigment epithelial (RPE) cells. METHODS: RPE cells were incubated with oxysterols (50 micro M of 24-hydroxycholesterol, 25-hydroxycholesterol, or 7-ketocholesterol) for 24 hr and 48 hr. Oxysterol content was determined in cells and in corresponding media by gas chromatography. Mitochondrial activity was measured by mitochondrial dehydrogenase activity. The intracellular formation of reactive oxygen species in RPE cells was detected by using the fluorescent probe DCFH-DA. IL-8 was assayed in the supernatants by ELISA, and the corresponding cellular transcripts were semiquantified by RT-PCR. RESULTS: Analyses of the oxysterols content in the RPE cells and corresponding media suggested a high rate of cellular uptake, although some differences were observed between 7-ketocholesterol on the one hand and 24-hydroxycholesterol and 25-hydroxycholesterol on the other hand. All oxysterols induced slight mitochondrial dysfunctions but a significant 2- to 4-fold increase in reactive oxygen species (ROS) production compared with the control. They also enhanced IL-8 gene expression and IL-8 protein secretion in the following decreasing order: 25-hydroxycholesterol > 24-hydroxycholesterol > 7-ketocholesterol. CONCLUSIONS: We conclude that in confluent primary porcine RPE cells, 24-hydroxycholesterol, 25-hydroxycholesterol, and 7-ketocholesterol are potent inducers of oxidation and inflammation.